ABSTRACT A great majority of compounds important for the treatment and study of human disease have their origin in natural products, many of which have cyclic structures that are critical for their biological activities. By exploiting the machinery by which these compounds are biosynthesized, it is possible to enhance or vary the biological activities of the parent molecules and apply the principles learned to new systems. However, in order to fully realize the potential of such an approach, the biosynthetic pathways of these compounds must be characterized and the underlying chemistry thoroughly understood. In this spirit, we have identified four systems to be investigated in the next funding period. The first specific aim is the mechanistic study of [4+2]-carbocyclases with the ultimate goal of identifying and definitively characterizing the first true Diels-Alderase. This is an emerging class of enzymes that have often proven challenging to study; however, we have already demonstrated that SpnF catalyzes a [4+2]-cycloaddition as its sole function during spinosyn A biosynthesis, and it will thus be the focus of this specific aim. Abycyc, a homologous [4+2]-carbocyclase in the abyssomicin pathway will also be studied for comparison. These reactions will be examined using kinetic isotope effect measurements, thermokinetic analyses, structural biology and computational methods to determine whether pericyclic transformations take place and how the cyclizations are accelerated by the enzymes. The second specific aim seeks to understand the biological origin of peptidyl nucleoside antibiotics (PNAs). We have recently uncovered strong evidence that the pyranose cores of these biologically active nucleosides are actually biosynthesized as polyketides thereby marking a significant departure from our current understanding of sugar biosynthesis. Through the collective application of our expertise in molecular biology, chemical synthesis and enzymology, we aim to investigate the biosynthesis of the PNAs amipurimycin and the miharamycins as a direct test of this hypothesis. The third specific aim focuses on the biosynthesis of unprecedented biochemical ring systems with the epoxyoxocin ring of nogalamycin and the annelated cyclobutane rings of the ladderane lipids serving as the principle natural products of interest. The epoxyoxocin ring of nogalamycin is a key contributor to its potency as a DNA intercalating agent; however, there is little information as to how this unique ring system is installed and thus how it could be reengineered for biomedical purposes. Likewise, the fused cyclobutanes of the ladderanes are believed to play an important role in anammox bacteria as part of the global nitrogen cycle. Nevertheless, it is unknown how these elaborate carbocycles are formed and genetic analyses imply the involvement of the exciting new class of cobalamin-dependent radical SAM enzymes. These research aims have been selected on the basis of their novelty, implications for the field of mechanistic enzymology, and potential utility in biomedical and biotechnological research at the basic and translational levels.